基于基因组挖掘策略的链霉菌活性天然产物发现

Discovery of bioactive secondary products from Streptomyces based on genome mining strategy

目的利用基因组挖掘策略在链霉菌中发现活性天然产物。方法首先,全测序获得马来西亚链霉菌(Streptomyces malaysiensis DSM 41697)全基因组序列。利用antiSMASH等在线工具对马来西亚链霉菌编码的潜在次级代谢产物生物合成基因簇进行初步预测。利用Blast、2ndFind等网站,确认基因簇中各基因的边界并对其功能进行注释,寻找目标基因簇。其次,尝试不同的发酵条件,并通过HPLC、LC-MS初步确定目标化合物是否产生。最后,分离和纯化目标化合物,并鉴定其结构。结果来自马来西亚链霉菌基因组的两个基因簇分别被命名为帕达酰胺生物合成基因簇和苯扎他汀生物合成基因簇。结合Blast和2ndFind软件分析,对基因簇进行边界确定和功能注释。通过尝试多种发酵条件,分离纯化获得了3种帕达酰胺类化合物和1种苯扎他汀类化合物。它们分别被鉴定为帕达酰胺A、帕达酰胺B、帕达酰胺A'和马来霉素。结论以基因组挖掘策略为指导,从微生物基因组中发现可能的天然产物生物合成基因簇,并通过多种发酵条件确定目标基因簇对应的产物是可行的。该策略为解决各类天然产物的来源及发现新结构天然产物提供了新思路,为活性化合物的寻找及其合成生物学奠定了基础。

Objective To discover active natural products from Streptomyces using a genome mining strategy.Methods Initially,the whole genome sequence of Streptomyces malaysiensis DSM 41697 was obtained through sequencing.The antiSMASH tool was utilized for the preliminary prediction of gene clusters responsible for potential secondary metabolite biosynthesis in Streptomyces malaysiensis.Subsequently,through the use of Blast,2ndFind,and other online tools,the target gene clusters were analyzed and the gene boundaries were confirmed.Following this,various fermentation conditions were tested to preliminarily determine the production of the target compounds using HPLC and LC-MS.Finally,the target compounds were isolated,purified,and their structures elucidated.Results Two biosynthetic gene clusters from Streptomyces malaysiensis were identified as padanamide synthesis and benzastatin biosynthesis,respectively.Through the analysis of online tools Blast and 2ndFind,the boundaries of the two clusters were confirmed,and all genes within the clusters were annotated.Subsequently,after optimizing the fermentation medium,three padanamide members and one benzastatin derivative,malaymycin,were purified.Conclusion Guided by the genome mining strategy,it is feasible to discover potential natural product biosynthetic gene clusters from microbial genomes and subsequently determine the products of the target gene clusters through fermentation optimization.This strategy showcases an efficient method for the directed discovery of novel bacterial natural products and may offer new biosynthetic pathways for combinatorial biosynthesis and synthetic biology.